Team:NAU-CHINA/RESULTS/Contribution
CONTRIBUTION
Introduction
For the contribution part of our project, we decided to do further research on the green fluorescent protein (GFP), which is commonly used as the reporter of a gene circuit. We chose the biobrick BBa_K608011 from the iGEM distribution kit, a pSB1C3 plasmid which contains a medium constitutive promoter, a medium RBS and GFP coding sequence, and transformed it into E.coli DH5α and E.coli BL21 to study the effect of temperatures and bacterial strains on the expression of GFP and the fluorescence intensity of bacteria. After that, we also extracted the GFP from the bacteria and identified the effect of pH on GFP activity.
Methods
The effect of temperature on E.coli DH5α and
E.coli BL21 containing BBa_K608011
This biobrick is presented in plasmid pSB1C3, which harbours a chloramphenicol resistant gene as a selection marker. So the Luria-Bertani (LB) plate and medium we used were supplemented with chloramphenicol (50 μg/ml).
BBa_K608011 was obtained from distribution kit and transformed into E.coli DH5α. Then the bacteria was coated on the LB plate, cultured overnight at 37 ℃. The next day we picked 4 colonies and incubated them in 5 mL LB medium for about 12 hours. The plasmids were extracted and transformed into E.coli BL21 and cultured overnight in 5 mL LB medium.
The overnight culture of E.coli BL21 and E.coli DH5α were diluted with fresh 150 mL LB medium and incubated at 37℃ to a 0.6 optical density at 600 nm (OD600), then separated into 3 groups, corresponding to the three temperature gradients 16℃, 30℃, 37℃.
The bacteria medium were incubated with shaking at 180 rpm and sampled every 2 hours and measured the OD600 and fluorescence (ex485, em520) data using 96 well microplate reader. LB media was served as control.
The effect of pH on GFP
We took out 50mL cell culture, centrifuged at 8000rpm for 10min, and discarded the supernatant. Then, add 10mL Tris-HCl buffer to the precipitate and re-suspended. The cells were crushed by ultrasonic for 10min. The supernatant was used as GFP crude protein solution.
0.5mL crude protein solution was added to 0.5mL buffer solution of pH 3 to 11 respectively, and incubated at 37℃ for 30min. Then we measured their fluorescence intensity using microplate reader. The crude protein solution added equal ddH2O was served as control.
Results
1.OD600 and fluorescence expression levels of E.coli BL21 and E.coli DH5α at 16℃, 30℃ and 37℃.
Fig.1 The effect of temperature on E.coli DH5α and E.coli BL21 containing BBa_K608011. A: OD600 values of bacteria at different temperatures; B: Florescence intensity of bacteria at different temperatures.
The figures showed that E.coli BL21 grew faster than DH5α. And the 37℃ was the most suitable temperature to grow basically.
Fig.2 Florescence intensity per OD600 of bacteria at different temperatures.
The data indicated that the fluorescence intensity of E.coli DH5α was higher than that of E.coli BL21 generally.
For E.coli DH5α, the fluorescence intensity at 16℃ was the lowest and the one at 37℃ was the highest in the first 2 hours. This is probably because cells in logarithmic growth phase, 37℃ is the optimal temperature for growth, and E.coli propagates fastest, so the expression of GFP was relatively high. After about 4 hours, the fluorescence intensity at 16℃is gradually higher than those at 30℃ and 37℃, that is because 16℃ is the optimal temperature for protein expression.
For BL21, the fluorescence intensity was highest at 37℃ comparatively.
The results can be used for reference in the study of GFP expression of these two stains of E.coli. Before the OD600 value of DH5α culture is up to 0.8, it can be cultured at 37℃ first to multiply, and then decrease to 16℃ to increase GFP expression. In this way, the total amount of GFP expression in E.coli DH5α medium can reach the optimal level. But for the BL21 stain, the temperature for cells’ growth can be maintained at 37℃. 16℃ and 30℃ are the most commonly used industrial fermentation temperatures, and 37℃ is the most commonly used temperature for bacterial culture. Characterization of BBa_K608011 at these three temperatures can be used as a guide for industrial and laboratory applications.
2.Fluorescence expression levels of GFP under different pH conditions
Fig.3 Florescence intensity of GFP at different pH.
The fluorescence intensity of blank control was similar to the one at pH 8. Surprisingly, the intensity of specimens at pH higher than 7 did not vary significantly from the intensity of which at pH 7, even higher, while the ones at lower pH deviated much more drastically.
Our experiment indicated that GFP could acquire higher fluorescence intensity at neutral or alkaline conditions. On the contrary, since acid has negative influence on GFP fluorescence intensity, we should pay attention to control the pH of cell culture medium higher than 7. Bacteria have a variety of secretion systems, and fluorescent proteins can also confirm the secretion efficiency of signal peptides. Once secreted, the protein is in an in vitro environment, so it is very important to characterize the nature of GFP in vitro environment. Our results suggested that GFP’s conformation can keep stable in alkaline conditions, which means it could be used in studying the functions of some proteins or polypeptides under extreme conditions. Our characterization is also useful for cell-free systems. On the contrary, since acid has negative influence on GFP fluorescence intensity, we should pay attention to control the pH of cell culture medium higher than 7.